A. Field of the Invention
The present invention relates generally to the field of biological molecules as components of coatings conferring an activity or other advantage to the coating proteinaceous molecule related to the biological molecule. More specifically, the present invention relates to proteins as such components of coatings. In one specific regard, the present invention relates to protein compositions capable of organophosphorus detoxification, and methods of reducing organophosphorus compounds on surfaces. More specifically, the present invention relates to coatings such as paints that degrade organophosphorus compounds such as pesticides and chemical warfare agents. The present invention further relates to paint and coating compositions and methods of their use to detoxify organophosphorus chemical warfare agents.
B. Description of the Related Art
Organophosphorus compounds (“organophosphate compounds” or “OP compounds”) and organosulfur (“OS”) compounds are used extensively as insecticides and are highly toxic to many organisms, including humans. OP compounds function as nerve agents. The primary effects of exposure to these agents are very similar, including inhibition of acetylcholinesterase and butyrylcholinesterase, with the subsequent breakdown of the normal operation of the autonomic and central nervous systems (Gallo and Lawryk, 1991).
Over 40 million kilograms of OP pesticides are used in the United States annually (Mulchandani, A. et al., 1999a). The number of people accidentally poisoned by OP pesticides has been estimated to be upwards of 500,000 persons a year (LeJeune, K. E. et al., 1998). Depending on the toxicity to the organism (e.g., humans), repeated, prolonged and/or low-dose exposure to an OP compound can cause neurotoxicity and delayed cholinergic toxicity. High-dose exposure can produces a fatal response (Tuovinen, K. et al., 1994).
Arguably of greater danger to humans, however, is the fact that some of the most toxic OP compounds are used as chemical warfare agents (“CWA”). Chemical warfare agents are classified into G agents, such as GD (“soman”), GB (“sarin”), GF (“cyclosarin”) and GA (“tabun”), and the methyl phosphonothioates, commonly known as V agents, such as VX and Russian VX (“R-VX” or “VR”). The most important CWAs are as follows: tabun (O-methyl dimethylamidophosphorylcyanide), which is the easiest to manufacture; sarin (“isopropyl methylphosphonofluoridate”), which is a volatile substance mainly taken up through inhalation; soman (“pinacolyl methylphosphonofluoridate”), a moderately volatile substance that can be taken up by inhalation or skin contact; cyclosarin (“cyclohexyl methylphosphonofluoridate”), a substance with low volatility that is taken up through skin contact and inhalation of the substance as a gas or aerosol; and VX (“O-ethyl S-diisopropylaminomethyl methylphosphonothioate”) and its isomeric analog R-VX [“O-isobutyl S-(2-diethylamino)-methylphosphonothioate, R-VX or VR”], both of which can remain on material, equipment and terrain for long periods, such as weeks, with R-VX being an especially persistent substance. All CWAs are colorless liquids with volatility varying from VX to sarin. VX is an involatile oil-like liquid, while sarin is a water-like, easily volatilized liquid. By addition of a thickener (e.g., a variety of carbon polymers), soman or other more volatile agents may be made to be less volatile and more persistent.
The CWAs are extremely toxic and have a rapid effect. Such agents enter the body through any of the following manners: inhalation, direct contact to the skin with a gas or with a contaminated surface, or through ingestion of contaminated food or drink. The poisoning effect takes longer when the agents enter through the skin, but is much faster when they are inhaled because of the rapid diffusion in the blood from the lungs. These toxins are fat-soluble and can penetrate the skin, but take longer to reach the deep blood vessels. Because of this, the first symptoms may not appear for 20-30 minutes after initial contact with a contaminated surface. This increases the danger for personnel entering a contaminated area, because the contamination may not be detected for 30 minutes or more (depending on concentrations) after the contaminated area is entered.
The first and most important method of protection from nerve agents is to prevent exposure. For military personnel and other first responders, masks and full body protective gear are available, but this equipment has certain drawbacks. Impermeable suits and even some air permeable suits are bulky and hot. The equipment inhibits free movement and tasks are harder and take longer to complete. In addition to those factors, hard physical work in these suits this may cause heat stress or even collapse. There may also be long delays before decontamination can be completed so the protective gear must be worn for long periods. This makes for a marginally acceptable first defense against a chemical warfare agent attack. Decontamination is also time-consuming so the equipment must often be destroyed and new equipment provided. It is also difficult to provide everyone with such protective equipment in the general population, and the effectiveness of such equipment diminishes during use. Tasks requiring detailed work using fingers and hands such as keystrokes on a keyboard, or pushing buttons on phones or equipment can be severely hampered by such bulky protective gear.
In addition to direct contact with a gaseous agent during an attack, surfaces that are exposed to the gas retain their toxicity for long periods of time. The OP nerve agents are soluble in materials such as paint, plastics, and rubber, allowing agents to remain in those materials and be released over long time periods. Nerve agents with thickening agents are even more persistent and difficult to decontaminate from a painted surface such as a wall, vehicle, or even a computer keyboard. It is understood that on painted metal surfaces, soman may persist for from one to five days, and that the less volatile VX may persist for 12 to 15 days. Under certain environmental conditions, OP compounds have been shown to persist indefinitely. On surfaces that are convoluted such as the surface of a military vehicle, the hidden surfaces that are less exposed to the environment can be especially difficult to decontaminate. Decontamination also requires detection, which is often not possible, and so resources and time may be wasted treating uncontaminated surfaces.
Historically, most approaches to chemical agent decontamination have focused on the treatment of surfaces after chemical exposure, whether real or merely suspected, has occurred. There are several current methods of decontamination of surfaces. One method is post-exposure washing with hot water with or without addition of detergents or organic solvents, such as caustic solutions (e.g., DS2, bleach) or foams (e.g., Eco, Sandia, Decon Green). Additional types of methods are an application of use of intensive heat and carbon dioxide applied for sustained periods, and incorporation of oxidizing materials (e.g., TiO2 and porphyrins) into coatings that, when exposed to sustained high levels of UV light, degrade chemical agents (Buchanan, J. H. et al., 1989; Fox, M. A., 1983). Chemical agent resistant coatings (“CARCs”) have been developed to withstand repeated decontamination efforts with such caustize and organic solvents. However, the resulting “decontaminated” materials are often still contaminated. Moreover, many decontamination procedures aerosolize contaminants on surfaces to be cleaned. In addition, it is often hard to clean certain kinds of surfaces such as those with rough texture, or with deep crevasses and other hard to reach areas that must often “self-decontaminate.”
Although each of these approaches can be effective under specific conditions, a number of additional limitations exist. Caustic solutions degrade surfaces, create personnel handling and environmental risks, and require transport and mixing logistics. Additionally, alkaline solutions, such as a bleaching agent, is both relatively slow in chemically degrading VX OPs and can produce decontamination products nearly as toxic as the OP itself (Yang, Y.-C. et al., 1990). While foams may have both non-specific biocidal and chemical decontamination properties, they require transport and mixing logistics, may have personnel handling and environmental risks, and are not effective on sensitive electronic equipment or interior spaces. CARCs have been shown to become porous after sustained UV light exposure that can create a sponge effect that may actually trap chemical agents and delay decontamination. Moreover, these approaches are not well suited for decontamination of convoluted surfaces. Decontamination with heat and carbon dioxide presents logistical requirements and does not allow rapid reclamation of equipment. UV-based approaches can be costly and have logistical requirements, including access to UV-generating equipment and power, as well as the production of toxic byproducts of degradation (Yang, Y.-C. et al., 1992; Buchanan, J. H. et al., 1989; Fox, M. A., 1983).
One attempted solution to the problem of surface contamination has been to provide paints with shedding (“chalking”) properties such as an acrylic surface that may shed, or at least not be penetrated by a CWA, making decontamination easier. This has been unsatisfactory solution, however, because the area remains contaminated and there is no way to know if the surface is or is not poisonous. In addition, shedding coatings over existing painted surfaces require additional materials and labor over a single coating. Shedding may or may not occur over timeframes necessary to protect personnel from residual nerve agents on contaminated surfaces, and in many instances may require washing despite the shedding characteristic.
Various enzymes have been identified that detoxify OP compounds, such as organophosphorus hydrolase (“OPH”), organophosphorus acid anhydrolase (“OPAA”), and DFPase, which detoxifies O,O-diisopropyl phosphorofluoridate (“DFP”). A number of civilian (e.g., Texas A&M University, private sector), and military laboratories [e.g., the Army research facilities at Edgewood (SBCCOM)] have worked on enzyme-based detection or decontamination systems for OP compounds. Various approaches taken in such laboratories include dispersion systems or immobilization systems of one or more OP degrading enzymes for use in detection or decontamination of OP compounds, as well as for convenience of handling of the enzyme preparation.
Sensors of OP compounds using an OP compound degrading enzyme have been described primarily for the detection of OP pesticides. OP compound sensors have been described that detect pH changes upon OP compound degradation using recombinant Escherichia coli cells expressing OPH cryoimmobilized in poly(vinyl)alcohol gel spheres (Rainina, E. I. et al., 1996). Endogenously expressed OPH from whole Flavobacterium sp. cells or cell membranes have been described as immobilized to glass membrane using poly(carbamoyl sulfonate) and poly(ethyleneimine) to produce a sensor of pH changes due to OP compound degradation (Gaberlein, S. et al., 2000a). OP compound sensors have been described that detect pH changes upon OP compound degradation using recombinant Escherichia coli cells, expressing OPH cytosolically or at the cell surface, that were fixed behind a polycarbonate membrane (Mulchandani, A. et al., 1998a; Mulchandani, A. et al., 1998b). An OP compound sensor has been described that detects optical changes upon OP compound degradation using recombinant Escherichia coli cells, expressing OPH at the cell surface, that were admixed in low melting point agarose and applied to membrane that was affixed to a fiber optic sensor (Mulchandani, A. et al., 1998c).
An OP compound sensor has been described that detects pH changes upon OP compound degradation using purified OPH chemically cross-linked with bovine serum albumin by glutaraldehyde on an electrode's glass membrane and covered with a dialysis membrane (Mulchandani, P. et al., 1999). Such chemically cross-linked OPH has been placed on a nylon membrane, and the membrane affixed to a fiber optic sensor to detect optical changes upon OP compound degradation (Mulchandani, A. et al., 1999a). Purified OPH has been immobilized by glutaraldehyde to glass-beads having aminopropyl groups in the construction of an OP compound degradation sensor (Mulchandani, P. et al., 2001a). An OP compound sensor has been described that detects optical changes upon OP compound degradation using recombinant Moraxella sp. cells, expressing OPH at the cell surface, that were admixed in 75% (w/w) graphite powder and 25% (w/w) mineral oil and placed into an electrode cavity (Mulchandani, P. et al., 2001b). Purified OPH was attached to silica beads by glutaraldehyde or N-γ-maleimidobutyrylozy succinimide ester linkages, and the beads placed as a layer on a glass slide to construct a sensor (Singh, A. K. et al., 1999). Purified OPH has been labeled with fluorescein isothiocyanate and absorbed to poly(methyl methacrylate) beads that were placed on a nylon membrane to construct a sensor that detects OP compound cleavage by decreased fluorescence (Rogers, K. R. et al., 1999). Purified OPH has been immobilized by placement within a poly(carbamoyl sulfonate) prepolymer that was allowed to polymerize on a heat-sealing film in the construction of a sensor (Gaberlein, S. et al., 2000b). A purified fusion protein comprising OPH and a FLAG octapeptide sequence was immobilized to magnetic particles (Wang, J. et al., 2001). Additional sensors using OPH have been described (Mulchandani, A. et al., 2001).
Different OP compound degrading enzyme compositions have been described, primarily for the detoxification of OP pesticides (Chen, W. and Mulchandani, A., 1998; LeJeune, K. E. et al., 1998a). A parathion hydrolase enzyme degrading cell extract has been immobilized onto silica beads and porous glass (Munnecke, D. M., 1979; Munnecke, D. M., 1978). OPH has also been immobilized onto porous glass and silica beads (Caldwell, S. R. and Raushel, F. M., 1991b). Purified OPH has been mixed with fire fighting foams in an attempt to create a readily dispersible decontamination composition (LeJeune, K. E., and Russell, A. J., 1999; LeJeune, K. E. et al., 1998b). Purified OPH has been incorporated into micelles in an OP compound degradation device (Komives, C. et al., 1994). Purified OPH has been encapsulated in a liposome for use in OP compound degradation (Pei, L. et al., 1994; Petrikovics, I. et al., 1999). OPH enzyme supported by glass wool in a biphasic solvent and gas phase reactor for OP compound detoxification has been described (Yang, F. et al., 1995). Purified OPH has also been immobilized onto trityl agarose and nylon (Caldwell, S. R. and Raushel, F. M., 1991a). Recombinant Escherichia coli cells co-expressing OPH and a surface expressed cellulose-binding domain have been immobilized to cellulose supports (Wang, A. A. et al., 2002). Partly purified OPH, acetylcholinesterase or butyrylcholinesterase has been incorporated into polyurethane foam sponges (Havens, P. L. and Rase, H. F., 1993; Gordon, R. K. et al., 1999). Partly purified or purified OPH has been incorporated into solid polyurethane foam (LeJeune, K. E. and Russell, A. J., 1996; LeJeune, K. E. et al., 1997; LeJeune, K. E. et al., 1999). Recombinant Escherichia coli cells expressing OPH have been immobilized in a poly(vinylalcohol) cryogel (Hong, M. S. et al., 1998; Efremenko, E. N. et al., 2002; Kim, J.-W. et al., 2002). Purified OPH has been immobilized in polyethylene glycol hydrogels (Andreopoulos, F. M. et al., 1999). Recombinant Escherichia coli expressing OPH at the cell surface has been immobilized to polypropylene fabric by absorption of the cells to the fabric (Mulchandani, A. et al., 1999b). Purified OPH was immobilized to mesoporous silica by Tris-(methoxy)carboxylethylsilane or Tris-(methoxy)aminopropylsilane (Lei, C. et al., 2002). A fusion protein comprising OPH and a cellulose-binding domain has been immobilized to cellulose supports (Richins, R. D. et al., 2000). Sonicated Escherichia coli cells expressing a fusion protein comprising OPH, a green fluorescent protein, and a polyhistidine sequence as an affinity tag, have been attached to a nickel-iminodiacetic acid-agarose bead resin (Wu, C.-F. et al., 2002). A fusion protein comprising OPH and a polyhistidine sequence as an affinity tag has been attached to a chitosan film (Chen, T. et al., 2001). A purified fusion protein comprising an elastin-like polypeptide and OPH has shown to reversibly bind to the hydrophobic surface of polystyrene plates at temperatures above 37° C. (Shimazu, M. et al., 2002).
In addition to OPH, other OP compound enzyme compositions have been described. Purified OPAA has been encapsulated in a liposome for use in OP compound degradation (Petrikovics, I. et al., 2000a; Petrikovics, I. et al., 2000b). Purified OPAA has been mixed with fire fighting foams, detergents, and a skin care lotion in an attempt to create a readily dispersible decontamination composition (Cheng, T.-C. et al., 1999). Purified squid-type DFPase has been encapsulated in erythrocytes for use in OP compound degradation (McGuinn, W. D. et al., 1993). Purified squid-type DFPase has been coupled to agarose beads (Hoskin, F. C. G. and Roush, A. H., 1982). Purified squid-type DFPase has also been incorporated into a polyurethane matrix (Drevon, G. F. et al., 2002; Drevon, G. F. et al., 2001; Drevon, G. F. and Russell, A. J., 2000).
US. Patent Publication no. US 2002/0106361 A1 discusses a marine anti-fungal enzyme for use in a marine coating. However, the substrate for the enzyme was incorporated into the marine coating, and the enzyme was in a marine environment as the organism from which it was obtained. Immobilized enzymes in an latex are discussed in the April, 2002 edition of “Emulsion Polymer Technologies,” by the Paint Research Association
However, to date, there has been limited success in using these and other approaches to harness the potential of these enzymes in systems that can be readily and cost effectively used in field-based military or civilian applications. Thus, despite the current understanding of the various OP compound degrading compositions and techniques, whether based on caustic chemicals or enzymes, there is a clear and present need for compositions and methods that can readily be used in OP compound degradation. This is particularly true for the detoxification of OP chemical warfare agents. In particular, compositions and methods are needed that will detoxify surfaces contaminated with OP compounds.